CN107475413A - One kind screening improves unrighted acid C20:The related SNP of the method for the long oyster of the contents of 3 Ω 6 primer pair - Google Patents
One kind screening improves unrighted acid C20:The related SNP of the method for the long oyster of the contents of 3 Ω 6 primer pair Download PDFInfo
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Abstract
The present invention relates to one kind to identify long oyster high unsaturated fatty acid eicosatrienoic acid (C20:3 Ω 6) the individual SNP marker of content is developed and its application process.By whole-genome association early stage, 38 and C20 on long No. 1 chromosome of oyster are identified:The significantly correlated cluster SNP signal of the contents of 3 Ω 6.The present invention is directed to region scaffold460_104,1 SNP site at 049 base, develops its corresponding detection method, the site has two kinds of base forms of A/T, and upstream and downstream 500bp nucleotide sequence is as shown in SEQ ID No.1.By the detection method, the close shellfish of AT genotype is screened, instructs oyster breeding.The invention provides carry out genotype identification, the method for raising filial generation unsaturated fatty acid content, the result is that the SNP marker obtained based on whole-genome association, effect are more stable to close shellfish before seed breeding.
Description
Technical field
The invention belongs to genetic engineering and genetic breeding field, is related to a kind of screening and improves unrighted acid C20:3
The related SNP of the method for the long oyster of the contents of Ω 6 primer pair.
Background technology
Oyster is a kind of important marine living resources, and one of most important sea-farming shellfish in the world.Although
China's oyster yield is very high, but the 3.3% of export volume Jin Zhan worlds oyster volume of trade, and average price is only other countries oyster
The 1/3 of product, this shows that China production oyster can only market one's own products mostly, it is difficult into international market and domestic high-end market.
Domestic oyster is the quality standard that product specification does not reach international trade the main reason for trace is difficult to find in international market in and, thing
This is also China's oyster culture industry overall efficiency for a long time low-level the main reason in reality.Therefore, high China to support
The economic benefit of oyster is grown, industry of realizing from the poorly efficient transition and upgrade to high yield and high efficiency of high yield, improves oyster product quality
The problem of being urgent need to resolve.
Polyunsaturated fatty acid is the important quality trait paid close attention in seashells, and important important meals
Eat aliphatic acid.The extra large oxygen invertebrate such as oyster is also with well-known rich in a variety of unrighted acids.Polyunsaturated fatty acid master
It is divided into two series of Ω -3 and Ω -6, Ω -6 series fatty acids belong to essential fatty acid, i.e., must be by being obtained in food, can not
Voluntarily synthesized in human body.So the content of Ω -6 polyunsaturated fatty acids in edible oyster is improved for improving oyster quality
And health has very important effect.And C20 in this project:3 Ω 6 belong to the serial unrighted acids of Ω -6
It is a kind of.
In China's aquatic livestock breeding research starting evening, traditional selection is mainly used at present, is hybridized, cycle length, slowly effect.
In the last few years, genomics developed, and the breeding technique such as molecular marking supplementary breeding and full-length genome selection has also obtained rapidly
Development, substantially increases that the genetic breeding of oyster is horizontal, not only makes it possible the breeding of complicated quality trait, while also will be aobvious
The Breeding Efficiency and precision of the lifting character of work.Blindness can be reduced using molecular marker assisted selection simultaneously, shortens breeding
The time limit, improve the efficiency of breeding.And molecular breeding is carried out, it is crucial to obtain with the significantly correlated SNP marker of character.And obtain at present
Obtaining the method for molecular labeling mainly includes the method for QTL positioning and whole-genome association.Although QTL positioning can quickly be determined
The position chromosome segment associated with character, but because its linkage analysis based on limited meiosis, the genome of positioning are big
The region of fragment is all chained together, it is difficult to be accurately positioned.And whole-genome association method can then overcome this shortcoming,
Population interconnection analysis is carried out to hereditary variation gene in the range of full-length genome, by associated bit point location in the section of very little,
Significantly improve the degree of accuracy and the precision of positioning, the heredity parsing for the complex character that is particularly suitable for use in.In recent years, as non-mode is given birth to
The completion of thing genome sequencing, positioned using GWAS technique study complexity Quantitative Trait Genes into fashion trend.
Although GWAS has played important function in the agro-ecology of crop and livestock and poultry is studied, in aquatic livestock also only
There is a small amount of report.And the method that the aquatic livestock reported at present, especially bivalve shellfish obtain SNP mainly uses candidate gene
Association analysis.But this method is to carry out the exploitation of pleomorphism site on known functional gene according to prior information, mark
Remember that relative number is few, differ and surely obtain main effect site, and there can not be one comprehensively to recognize the genetic regulation mechanism of phenotype
Know.With the completion of long oyster genome sequencing and the acquisition of high density genetic linkage mapses so that carry out GWAS analyses
It is possibly realized.The present invention is based on long oyster genome, and carrying out full-length genome by two generation sequencing technologies resurveys sequence, and is directed to insatiable hunger
Whole-genome association is carried out with content of fatty acid, obtains the main effect pleomorphism site and key gene for controlling its content.With
The SNP site developed in the past is compared, and the SNP signal confidence level that the present invention obtains is higher, and suited-community scope is wider, more effectively should
For carrying out the screening of unsaturated fatty acid content.
The content of the invention：
It is an object of the invention to provide a kind of and long oyster unrighted acid C20:The related SNP marker of the contents of 3 Ω 6,
Molecular marker assisted selection for long oyster provides reference.
Specific acquisition SNP methods are as follows：(1) experiment material collection and homogeneity raise and train：It is wild to collect 486 long oyster
Raw individual, carries out homogeneous cultivation.(2) measure of phenotypic data：C20 is carried out to long oyster individual using gas chromatography:3Ω
6 assays.(3) Genotyping：Using two generation sequencing technologies platforms, 486 oyster parents are carried out to resurvey sequence, examination SNPs
Site simultaneously carries out individual parting, obtains effective SNPs sites for association analysis, builds the haplotype collection of illustrative plates of oyster.(4) close
Connection analysis：Whole-genome association is carried out using mixed linear model, obtains 38 SNP sites significantly associated with character
(P-value<10-6), in the range of long oyster genome scaffold 460 15,302-114,852kb.Pass through LD
Block is analyzed, 38 SNP site close linkages (LD ＞ 0.7).The Manhattan figure such as accompanying drawing 1 that whole-genome association obtains
It is shown.Then choose and be located at scaffold460_104, the base on 049kb, P-value is 5.53 × 10-10, as subsequently opening
The SNP site of hair.Two base forms of A and T be present in the site.In this research, positioned at the 15,302-114 of scaffold 460,
Other SNP sites in the range of 852kb all apply identical authentication method.The present invention is achieved by the following technical programs：
Long oyster genomic DNA is template, using special primer, carries out peripheral amplification:
Sense primer F：5’-ACGTCACAAGTAGGTGTTTAGAGA-3’
Anti-sense primer R：5’-CTTTTCACGTCTCGCCATTCC-3’.
SNP marker is located at long No. 1 chromosome scaffold460_104,049 base of oyster genome；A be present in the site
With two base forms of T, SNP marker loci gene type identification, different genotype C20 are carried out to close shellfish before seed breeding:3
The order of the contents of Ω 6 is AT>TT>AA, selected according to the close shellfish of the SNP marker site different genotype, by screening the site
The close shellfish of AT genotype, improve the C20 of progeny population:The contents of 3 Ω 6；It is as follows including step：
1) long oyster genomic DNA is extracted, dilutes 10-20ng/uL using aqua sterilisa or TE buffer solutions；Using specifically drawing
Thing, carry out peripheral amplification:2) it is template to take above-mentioned long oyster genomic DNA, and reaction system is prepared using special primer F and R：
Sense primer F：5’-ACGTCACAAGTAGGTGTTTAGAGA-3’
Anti-sense primer R：5’-CTTTTCACGTCTCGCCATTCC-3’；
Genomic DNA 1uL, universal PC R mix 5uL, primers F and each 0.2uL of R, sterilizing distilled water 3.6uL；It is described anti-
System is answered to amplify on year-on-year basis；
3) response procedures of PCR amplifications are：
4) prepared by SnaPshot templates：
37 DEG C of 5U SAP and 2U ExoI concussions mixing is added in 15 μ L PCR primers and is incubated 1 hour, then 75 DEG C of insulations
15min is to inactivate SAP and ExoI enzymes；
5) SnaPshotPCR amplifications and purifying：
Specific primer sequences are：
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGACCGAACTTTATGAACT-3’
Product purification：In the above-mentioned SNaPshot templates of 10 μ L add 1U SAP, concussion mix, 37 DEG C insulation 1hr, 75 DEG C
15min is incubated with inactivator, 4 DEG C can preserve 24 hours or -20 DEG C long-term preserve；
6) Capillary Electrophoresis；
It is prepared by electrophoresis Sample：First by 20 times of SNaPshot product dilutions：
Reagent scope is：The μ L of 0.25 μ L, SnaPshot product of Hi-Di Formamid9.25 μ L, GS-120LIZ 0.5,
95 DEG C of denaturation 5min, rear rapid ice-cold 4min；
Capillary Electrophoresis：Capillary Electrophoresis is carried out to the sample prepared using 3730XLDNA Analyzer and collects letter
Number；Environmental condition：Laboratory temperature：18-25℃；Capillary pipe length：50cm；Furnace temp：60℃；Working voltage：15kV；
As a result experimental result is analyzed using GeneMapper V4.0；Judge different genotype.
Product purification：1U SAP are added in the above-mentioned SNaPshot PCR primers of 10 μ L, concussion mixes, 37 DEG C of insulation 1hr,
With inactivator, 4 DEG C can preserve 24hr or -20 DEG C long-term and preserve 75 DEG C of insulation 15min.
5th, Capillary Electrophoresis.
1) prepared by electrophoresis Sample：First by 20 times of SNaPshot product dilutions：
95 DEG C of denaturation 5min, rear rapid ice-cold 4min.
2) Capillary Electrophoresis.Capillary Electrophoresis is carried out to the sample prepared using 3730XLDNA Analyzer and collected
Signal.Environmental condition：Laboratory temperature：18-25℃；Capillary pipe length：50cm；Furnace temp：60℃；Working voltage：
15kV.As a result experimental result is analyzed using GeneMapper V4.0.Judge different genotype.
With long oyster unrighted acid C20:The potential application of the related SNP marker of the contents of 3 Ω 6：Up to the present, remove
Outside this patent, in oyster, there has been no the report based on whole-genome association exploitation SNP marker.This result of study is relative
In the SNP marker developed in the past, confidence level is higher, and the scope of suited-community is wider, and effect is more stable.Before seed breeding, lead to
Non-lethality sampling extraction oyster genomic DNA is crossed, judges close shellfish base using SNP marker and its authentication method as described above
Because of type, the long oyster unrighted acid C20 of offspring is effectively improved by screening GG genotype parent shellfishes:The contents of 3 Ω 6.
Brief description of the drawings
Fig. 1 is C20:The Manhattan figure of the content whole-genome associations of 3 Ω 6.
Embodiment：
Further explain the present invention with reference to embodiments, but embodiment does not do any type of limit to the present invention
It is fixed.
Embodiment 1：
A) collection of sample：100 individuals of wild population of seedling are incubated in collection Jiangnan simultaneously, and it is dissected, takes closed shell
Flesh and remaining tissue, are saved backup with liquid nitrogen flash freezer after -80 DEG C.
B) DNA extraction：Extract the genomic DNA of 100 samples and use Uv-spectrophotometric Determination concentration, according to
Genomic DNA is diluted to 10-20ng/uL by measure concentration using aqua sterilisa；
C) SNP site genotype detection is carried out using SnaPshot：(1) special primer is utilized, carries out peripheral amplification.Take
It is template to state long oyster genomic DNA, and reaction system is prepared using primers F and R：Genomic DNA 1uL, universal PC R mix
5uL, primers F and each 0.2uL of R, sterilizing distilled water 3.6uL；The reaction system can amplify on year-on-year basis；Primer sequence is：
Sense primer F：5’-ACGTCACAAGTAGGTGTTTAGAGA-3’
Anti-sense primer R：5’-CTTTTCACGTCTCGCCATTCC-3’
PCR amplification response procedures be：
72℃0-10min
(2) prepared by SnaPshot templates.5U SAP and 2U ExoI concussions are added in 15 μ L PCR primers and mix 37 DEG C of guarantors
Then warm 1hr is incubated 15min to inactivate SAP and ExoI enzymes for 75 DEG C.
(3) SnaPshotPCR amplifications and purifying.Template using PCR primer as SNaPshot PCR, after purifying is good,
It is every kind of respectively to take 2 μ L to mix.SNaPshot PCR are concretely comprised the following steps：
Primer sequence is：
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGACCGAACTTTATGAACT-3’
Product purification：1U SAP are added in the above-mentioned SNaPshot PCR primers of 10 μ L, concussion mixes, 37 DEG C of insulation 1hr,
With inactivator, 4 DEG C can preserve 24hr or -20 DEG C long-term and preserve 75 DEG C of insulation 15min.
(4) Capillary Electrophoresis.It is prepared by electrophoresis Sample：First by 20 times of SNaPshot product dilutions：
95 DEG C of denaturation 5min, rear rapid ice-cold 4min.The sample prepared is carried out using 3730XLDNA Analyzer
Capillary Electrophoresis simultaneously collects signal.Environmental condition：Laboratory temperature：18-25℃；Capillary pipe length：50cm；Furnace temp：
60℃；Working voltage：15kV.As a result experimental result is analyzed using GeneMapper V4.0.Judge different genotype.
D) interpretation of result:After detection, only 9 idiotypes are A/A, and 33 idiotypes are T/A, 45 individuals
Genotype is T/T, and 13 idiotypes are NN,
E) unrighted acid C20:The detection of the contents of 3 Ω 6：Data are shown in figure, different genotype C20:The contents of 3 Ω 6
Order be AT>TT>AA.AT genotype C20:The contents of 3 Ω 6 are compared to AA and TT types, C20:The contents of 3 Ω 6 significantly improve 47%
With 11%.So the parting and C20 in the site:The contents of 3 Ω 6 significantly associate., can by screening CC types individual in breeding
To significantly improve offspring C20:The contents of 3 Ω 6.
Table 1：288 individual unrighted acid C20:The contents of 3 Ω 6 and Genotyping analysis.
Sequence table (1) SEQ ID NO.1 information sequence characteristic length：1001bp, scaffold1597_36,675 site
Upstream and downstream 500bp.
Type：Nucleic acid
Chain：It is single-stranded
Topological structure：It is linear
Molecule type：DNA
Source：Long oyster
Sequence description：
CATCACATTGTAAGCGGTTTTATTGGCGAAGATCAGAGATGATAATAAGGAAAGGTCAATCATTTCTTCTTAAGGAC
TGAAGCAAAAGACTCCGGACTTCGCGCATATAACCGATACACAATTATTAAGCTATTTTATAAGTGAAAGTCATATA
AATAAAGTAATTGTGATTGACACTTGGATTATCAAAACCTGCGGTATATTACATATATCTATAAACACCATAAAATA
CGCTCAAACTAAAATTACCGATGTAATTACAGGCAAAATACAGAAAACATAATAGTTTTATGTCTACGTCACAAGTA
GGTGTTTAGAGACAATGCTACTATCGACAGCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGTGCAAATCTC
TCTTAATTAATTAAAACATTACAAATGATTTGCCATTTGTAACACCATCTTTGCTTTTTGTAGAAATCATTTCTTTC
ACACCTTTACCGACAGCGGGGACCGAACTTTATGAACTAAATGTGTTGTTAACTTACCAAAACTTTTCAAGTTTTCT
CGAGACAGCATTGTTCGAATGAAAAAGCGGTAGGAATGGCGAGACGTGAAAAGTTTCTTTTTTCTGTAAAATAATGT
TGTATTTAAAAATCTTTCTATTGGTATAACAAAGACAATATTTTAAAAGAAAATCCGAGGTTGTTATTTCAAATTGA
CACGATATTGTTCCAAACACGAAGCAATTTCTTGTATCAACAGTGATGTAGCAAGATAACAGATCGTACATCGACAA
AACGATTAACATCAATTAAATTCACTCGTTCATAAAATGTTTCAATCAAAAGGGATATTCTGTATGTATTGGTGAAG
ATATTAGCACAGAATGACTATTACATTGTGTCATTAATCTTATAAATATTTGATTTTGGTCTTAAAAGGAATGTGTA
GGAAATTACGTTTAAAGTTTCATACCCGAAAATGTAAACGGTTAATTTAAAAACATTTAACAGCCTGTGACCGCGGA
。
The present invention by whole-genome association early stage, identify 38 on long No. 1 chromosome of oyster with
C20:The significantly correlated cluster SNP signal of the contents of 3 Ω 6.The present invention is at the scaffold460_104,049 bases of the region
1 SNP site, develops its corresponding detection method, and the site has two kinds of base forms of A/T, upstream and downstream 500bp nucleosides
Acid sequence is as shown in SEQ ID No.1.By the detection method, the close shellfish of AT genotype is screened, instructs oyster breeding.The present invention
Provide and genotype identification is carried out to close shellfish before seed breeding, the method for improving filial generation unsaturated fatty acid content, its result
It is the SNP marker obtained based on whole-genome association, effect is more stable.
Sequence table
<110>The Institute of Oceanology of the Chinese Academy of Sciences
<120>One kind screening improves unrighted acid C20:The related SNP of the method for the long oyster of the contents of 3 Ω 6 primer pair
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1001
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(1001)
<400> 1
catcacattg taagcggttt tattggcgaa gatcagagat gataataagg aaaggtcaat 60
catttcttct taaggactga agcaaaagac tccggacttc gcgcatataa ccgatacaca 120
attattaagc tattttataa gtgaaagtca tataaataaa gtaattgtga ttgacacttg 180
gattatcaaa acctgcggta tattacatat atctataaac accataaaat acgctcaaac 240
taaaattacc gatgtaatta caggcaaaat acagaaaaca taatagtttt atgtctacgt 300
cacaagtagg tgtttagaga caatgctact atcgacagct ctctctctct ctctctctct 360
ctctctctct ctctgtgcaa atctctctta attaattaaa acattacaaa tgatttgcca 420
tttgtaacac catctttgct ttttgtagaa atcatttctt tcacaccttt accgacagcg 480
gggaccgaac tttatgaact aaatgtgttg ttaacttacc aaaacttttc aagttttctc 540
gagacagcat tgttcgaatg aaaaagcggt aggaatggcg agacgtgaaa agtttctttt 600
ttctgtaaaa taatgttgta tttaaaaatc tttctattgg tataacaaag acaatatttt 660
aaaagaaaat ccgaggttgt tatttcaaat tgacacgata ttgttccaaa cacgaagcaa 720
tttcttgtat caacagtgat gtagcaagat aacagatcgt acatcgacaa aacgattaac 780
atcaattaaa ttcactcgtt cataaaatgt ttcaatcaaa agggatattc tgtatgtatt 840
ggtgaagata ttagcacaga atgactatta cattgtgtca ttaatcttat aaatatttga 900
ttttggtctt aaaaggaatg tgtaggaaat tacgtttaaa gtttcatacc cgaaaatgta 960
aacggttaat ttaaaaacat ttaacagcct gtgaccgcgg a 1001
Claims (2)
1. one kind screening improves unrighted acid C20:The related SNP of the long oyster of the contents of 3 Ω 6 primer pair, it is characterised in that：
Long oyster genomic DNA is template, using special primer, carries out peripheral amplification:
Sense primer F：5’-ACGTCACAAGTAGGTGTTTAGAGA-3’
Anti-sense primer R：5’-CTTTTCACGTCTCGCCATTCC-3’.
2. the screening described in a kind of claim 1 improves unrighted acid C20:The method of the long oyster of the contents of 3 Ω 6, its feature
It is：
SNP marker is located at long No. 1 chromosome scaffold460_104,049 base of oyster genome；A and T be present in the site
Two base forms, SNP marker loci gene type identification, different genotype C20 are carried out to close shellfish before seed breeding:3Ω6
The order of content is AT>TT>AA, selected according to the close shellfish of the SNP marker site different genotype, by screening site AT bases
Because of the close shellfish of type, the C20 of progeny population is improved:The contents of 3 Ω 6；It is as follows including step：
1) long oyster genomic DNA is extracted, dilutes 10-20ng/uL using aqua sterilisa or TE buffer solutions；Using special primer, enter
The amplification of row periphery:2) it is template to take above-mentioned long oyster genomic DNA, and reaction system is prepared using special primer F and R：
Sense primer F：5’-ACGTCACAAGTAGGTGTTTAGAGA-3’
Anti-sense primer R：5’-CTTTTCACGTCTCGCCATTCC-3’；
Genomic DNA 1uL, universal PC R mix 5uL, primers F and each 0.2uL of R, sterilizing distilled water 3.6uL；The reactant
System can amplify on year-on-year basis；
3) response procedures of PCR amplifications are：
4) prepared by SnaPshot templates：
37 DEG C of 5U SAP and 2U ExoI concussions mixing is added in 15 μ L PCR primers and is incubated 1 hour, then 75 DEG C of insulations
15min is to inactivate SAP and ExoI enzymes；
5) SnaPshotPCR amplifications and purifying：
Specific primer sequences are：
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGACCGAACTTTATGAACT-3’
Product purification：1U SAP are added in the above-mentioned SNaPshot templates of 10 μ L, concussion mixes, 37 DEG C of insulation 1hr, 75 DEG C of insulations
For 15min with inactivator, 4 DEG C can preserve 24 hours or -20 DEG C long-term preserve；
6) Capillary Electrophoresis；
It is prepared by electrophoresis Sample：First by 20 times of SNaPshot product dilutions：
Reagent scope is：Added in 10 μ L deionized waters, μ L, GS-120LIZ0.25 the μ L of Hi-Di Formamid 9.25,
The μ L of SnaPshot products 0.5；
95 DEG C of denaturation 5min, rear rapid ice-cold 4min；
Capillary Electrophoresis：Capillary Electrophoresis is carried out to the sample prepared using 3730XLDNA Analyzer and collects signal；
Environmental condition：Laboratory temperature：18-25℃；Capillary pipe length：50cm；Furnace temp：60℃；Working voltage：15kV；Knot
Fruit is analyzed experimental result using GeneMapper V4.0；Judge different genotype.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588242A (en) * | 2018-07-11 | 2018-09-28 | 浙江万里学院 | A kind of SNP site of long oyster AHR genes |
| CN112626238A (en) * | 2021-01-19 | 2021-04-09 | 浙江万里学院宁海海洋生物种业研究院 | Characteristic sequence for identifying crassostrea sikamea, specificity identification primer and identification method |
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| CN113793637A (en) * | 2021-09-06 | 2021-12-14 | 中国科学院水生生物研究所 | Whole genome association analysis algorithm based on parental genotype and progeny phenotype |
| CN113793637B (en) * | 2021-09-06 | 2022-07-26 | 中国科学院水生生物研究所 | Whole genome association analysis method based on parental genotype and progeny phenotype |
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